List of Prior Art Literature
    Stewart, S. J. 1991. Francisella. In: Balows, A., W. J. Hausler, Jr., K. L. Herrmann, H. D. Isenberg, and H. J. Shadomy (ed.). Manual of Clinical Microbiology. Am. Society for Microbiology, pp. 454-456.    Franz, D. R., P. B. Jahrling, A. M. Friedlander, D. J. McClain, D. L. Hoover, W. R. Bryne, J. A. Pavlin, G. W. Christopher and E. M. Eitzen, Jr. 1997. Clinical recognition and management of patients exposed to biological warfare agents. JAMA, 278:399-411.    Evans, M. E., and A. M. Friedlander. Tularemia. In: F. R. Sidell, E. T. Takafuji and D. R. Franz (ed.) Medical Aspects of Chemical and Biological Warfare. 1997. Published by the Office of the Surgeon General at TMM Publications. pp. 503-512.    Cherwonogrodzky, J. W., M. H. Knodel, and M. R. Spence. 1994. Increased encapsulation and virulence of Francisella tularensis live vaccine strain (LVS) by subculturing on synthetic medium. Vaccine. 2:773-775.    Corbel, M. J. Recent advances in the study of Brucella antigens and their serological cross-reactions. 1985. Veterinary Bulletin. 55: 927-942.    Sjostedt, A. 1997. Host-parasite interactions during tularemia. (Introductory remarks to the presentation, given at the Medical Protection B Conference, Munich, Germany).    Golovliov, I., M. Ericsson, L. Akerblom, G. Sandstrom, A. Tarnvik and A. Sjostedt. 1995. Adjuvanicity of ISCOMS incorporating a T-cell reactive lipoprotein of the facultative intracellular pathogen Francisella tularensis. Vaccine. 13:261-267.    Hood, A. M. 1977. Virulence factors of Francisella tularensis. J. Hyg. Camb. 79: 47-60    Ancuta, P., T. Pedron, R. Girard, G. Sandstrom and R. Chaby. 1996. Inability of the Francisella tularensis lipopolysaccharide to mimic or to antagonize the induction of cell activation by endotoxins. Infect. Immun. 64: 2041-2046.    Conlan, J. W., H. Shen, A. Webb, M. B. Perry. 2002. Mice vaccinated with the O-antigen of Francisella tularensis LVS lipopolysaccharide conjugated to bovine serum albumin develop varying degrees of protective immunity against systemic or aerosol challenge with virulent type A and type B strains of the pathogen. Vaccine 20: 3465-3471.    Cherwonogrodzky, J. W. 1983. Factors controlling haemolysin production in Vibrio parahaemolyticus. Ph.D. thesis, University of Toronto, pages 161-162.
Tularemia is primarily a disease of wildlife that spreads to humans incidentally such as by insect or tick bites, handling infected carcasses or by drinking contaminated water. The disease usually progresses from an ulcer (at the site of infection or within the bowel if ingested) to oculoglandular infections (eyes are stressed and ‘flu’-like symptoms such as chills, fever, headache and general aches and pains becoming progressively worse) then systemic gastrointestinal or pleuropneumonia tularemia that causes severe illness with a high mortality rate (30-60%) unless antibiotic therapy is given. Although the incidence of tularemia has declined with the decline of market hunting and trapping, it is still widespread around the globe, infecting wildlife, domestic animals and humans (Stewart, 1991). The bacterium is readily grown on simple medium with a cysteine supplement, and it is highly virulent when delivered as an aerosol or in contaminated water. It is a potential threat agent for biological warfare or terrorist programs (Franz et al., 1997).
With regards to medical countermeasures against tularemia, antibiotics can clear the infection, but the success of these antibiotics depends on where the infection has located and how early the patient is treated. A F. tularensis live vaccine strain (LVS) is available, but it has an IND (Investigational New Drug) status, its efficacy against exposures by different routes of infection is questionable (Evans and Friedlander, 1997) and under certain conditions it appears to revert to its virulent parental form (Cherwonogrodzky et al., 1994). There is therefore a need for both a new, more effective and safe vaccine, a means of assessing the stability of different batches of the existing LVS vaccine, and for assessing by a simple blood test if a vaccinate is indeed protected from tularemia. As Brucella and F. tularensis cross-react (Corbel, 1985), it would benefit serodiagnosis if an antigen could determine which bacterium had infected a patient.
In theory, it might be viewed that sera from either vaccinated or infected animals or humans might have antibodies that could identify components key to the disease process and hence potentially useful as vaccine candidates. To date this has not happened. For the first part, vaccination often gives limited results (e.g. although the LVS protects against tularemia, its efficacy for protecting against infection by different routes is questionable) with antibody titers either being low or rapidly diminishing with time. For the second part, infected animals or human have an illness where the immunity has either failed or has responded incorrectly to what was required for protection. Indeed, Edward Francis (from whom Francisella is named) had 3 infections of tularemia and eventually died from this disease (Sjostedt, 1997). An infection did not provide Francis with immunity. Although sera from vaccinated or infected patients do have antibodies with affinity for low molecular proteins of F. tularensis, these proteins do not appear to give protection when used as vaccines (Golovliov et al., 1995). The approaches used by the experts in the field to date teach away from novel approach of the present invention on how to identify potentially useful components.
In a previous publication (Cherwonogrodzky et al., 1994), the capsule of F. tularensis was viewed as a virulence factor. However, in subsequent studies it was found that this was not the case. Mice did not become ill when they were infected with F. tularensis live vaccine strain subcultured more than 6 times in a synthetic salts medium, even though these bacteria still had extensive capsules. Evidently the initial speculation on the role of capsules for virulence of the bacterium was incorrect and teaches away from the later findings.
With regards to toxins, the current state of knowledge is that toxins are not present for F. tularensis (Hood, 1977; Ancuta et al., 1996). In unpublished studies, we found that when the third (broth or agar) subculture had the supernatant filter-sterilized (0.2 μm filter used), and 0.05 ml was given to mice intranasally, initially the mice appeared normal. This initial observation appeared to confirm the existing state of knowledge that no toxins were present in F. tularensis cultures. However, 24 hours after the supernatant inoculation, all mice were found dead in their cages. These latter (unreported) results proved, contrary to the literature, that a toxic agent was being produced by the bacterium but that it had a delayed action on the test animals.